Haemophilus influenzae is a common cause of localized respiratory tract disease, including otitis media, sinusitis, bronchitis, and pneumonia. Less commonly, this organism causes serious systemic disease, such as meningitis, septicemia, and endocarditis. The initial step in the pathogenesis of H. influenzae disease involves colonization of the upper respiratory mucosa. We have identified a non-pilus adhesin called Hia, which is present in nontypable (nonencapsulated) H. influenzae and promotes high-affinity attachment to human epithelium. In addition, we have identified a homolog of Hia called Hsf, which is present in nearly all encapsulated strains of H. influenzae and also mediates high-affinity adherence to epithelial cells. Both Hia and Hsf are members of the expanding family of auto-transporter proteins, characterized by an N-terminal signal peptide, an internal passenger domain, and a C-terminal trans-locator domain. In contrast to classic auto-transporters, Hia and Hsf have a translocator domain that is unusually short and forms a trimer. For both Hia and Hsf, the passenger domain has two distinct binding regions that interact with the same host cell receptor, although with differing affinities. We have crystallized a polypeptide containing the Hia primary binding domain and find a densely packed trimer with three-fold symmetry and three identical major grooves, acidic pockets, and globular domains. In Aim 1 of this proposal, we will define the surfaces of the Hia and Hsf binding domains involved in interaction with host cells. In addition, we will explore whether trimer formation is required for adhesive activity. In Aim 2, we will characterize the structure and mechanism of action of the Hia and Hsf translocator domain. In Aim 3, we will identify the Hia/Hsf host cell receptor and elucidate the host cell response to Hia and Hsf-mediated adherence. From a practical perspective, the results of these experiments may facilitate efforts to develop a vaccine protective against non-type b H. influenzae and suggest targets for novel antimicrobials with activity against a broad range of gram-negative bacteria. More generally, they may provide fundamental insights into host-pathogen relations, the autotransporter family of proteins, and protein secretion.